幽门螺杆菌尿素酶B亚单位在大肠杆菌中的融合表达与鉴定

Fusion expression and detection of ureB from Helicobacter pylori in E.coli

目的 通过对幽门螺杆菌(Helicobacter pylori,Hipylori)尿素酶B亚单位在大肠杆菌中表达的研究,探索其免疫反应性。方法 用高保真PCR方法扩增H.pylori ureB基因,并定向克隆入pNEB193中,然后经酶切回收后插入原核表达载体pMAL-C2X进行融合表达。采用蛋白印迹法对表达产物进行鉴定。结果 特异PCR和酶切鉴定证实融合基因ureB克隆入表达载体,SDS-PAGE结果显示ureB在大肠杆菌中以融合蛋白形式MBP-ureB高效表达,约占细胞总蛋白量的26.7％。该融合蛋白可与H.pylori阳性病人血清发生特异反应。结论 成功构建了能高效表达H.pylori ureB的重组菌,为Hp基因工程疫苗的进一步研究奠定了基础。

Objective To research the expression of urease B subunit (ureB) of H. pylori and its ability to react with specific antigen. Methods The ureB gene were amplified from H. pylori by PCR and cloned directionally into vector pNEB193 .This gene was digested by using two kinds of restriction enzyme separately and cloned into the fusion expression vector pMAL-C2X,and the recombinant plasmid was then transform E. coli TB1 .The positive clones were screened by PCR and restriction enzyme digestion. The recombinant fusion protein MBP-ureB was induced to express by IPTG and was analyzed by SDS-PAGE and Western blot analysis. Results The ureB gene could be amplified from the recombinant fusion expression plasmid by PCR, and also the ureB gene fragment could be produced from these plasmids after restriction enzyme digestion. SDS-PAGE and optical density scanning indicated that the fusion protein was expressed in the recombinant vaccine strain TB1 (pMUB)as a protein with 105KD of molecular weight and was 26.7% of the total bacterial protein.The fusion protein could react with H. pylori ( + ) patient serum. Conclusions A recombinant vaccine strain expression fusion protein MBP-ureB of H. pylori was constructed and identified successfully, and this study will help to develop gene recombinant vaccine against H. pylori infection.